Development and Validation of RP HPLC method for the estimation of Etoricoxib in bulk and tablets.

Objective: Objective of the present analytical research work was to develop and validate Reverse Phase High Performance Liquid Chromatographic method (RP-HPLC Method) for the Etoricoxib in bulk and tablets dosage form. Methods: A RP-HPLC method has been developed and validated for estimation of ETOR in pharmaceutical oral dosage form. Method A (RP-HPLC Method): The RP-HPLC Method for Etoricoxib was developed using Shimadzu HPLC, LC-10, temperature maintained 25 0C, phenorex Gemini C18 (250 mm 4.60 mm 5?m), as stationary particle, isocratic mode. Water: ACN: OPA: TEA (40:60:0.1:0.1, v/vv/v). Mobile phase was maintained at a flow rate of 1.0 ml/min and detection was carried out at 245 nm. Results: Etoricoxib was found to be linear in the concentration range of 8 - 12 ?g/ml for RP-HPLC method. Retention time was found to be 2.7 min for Etoricoxib. The amount of Etoricoxib in marketed formulation was found to be 99.14 %. Interpretation and Conclusion: Results of assay and validation study were found to be satisfactory. So, the method can be successfully applied for the routine analysis of Etoricoxib

Development and Validation of UV Spectrophotometric and RP HPLC method for the estimation of Eszopiclone bulk and tablets.

Objective: Objective of the present analytical research work was to develop and validate Spectrophotometric method and Reverse Phase High Performance Liquid Chromatographic method (RP-HPLC Method) for the Eszopiclone bulk and tablets dosage form. Methods: A spectrophotometric method and a RP-HPLC method have been developed and validated for estimation of ESZ in pharmaceutical oral dosage form. Method A (RP-HPLC Method): The RP-HPLC Method for Eszopiclone was developed using Shimadzu HPLC, LC-10, temperature maintained 25 0C, phenorex Gemini C18 (250 mm 4.60 mm 5?m), as stationary particle, isocratic mode. MeOH: Water (80:20v/v) as mobile phase. Mobile phase was maintained at a flow rate of 1.0 ml/min and detection was carried out at 305 nm. Method B (UV SPECTROMETRY Method): The stock and working standard solutions of the drugs were prepared in methanol. Standard solutions were scanned over the range of 400-200 nm in spectrum mode of spectrophotometer at medium scanning speed using UV spectrophotometer 2450, SHIMADZU. The maximum absorbance for Eszopiclone was found at 305 nm. Both the methods were validated in accordance with ICH guidelines Results: Eszopiclone was found to be linear in the concentration range of 4 - 24 ?g/ml for spectrophotometric method and 5-30 ?g/ml for RP-HPLC method. Retention time was found to be 5.38 min for Eszopiclone. The amount of Eszopiclone in marketed formulation by spectrophotometric method was found to be 100.02 %, the amount of Eszopiclone in marketed formulation by RP-HPLC method was found to be 100.03 %. Interpretation and Conclusion: Results of assay and validation study were found to be satisfactory. So, the methods can be successfully applied for the routine analysis of Eszopiclone

A NEW REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE SIMULTANEOUS ESTIMATION OF SERRATIOPEPTIDASE AND DICLOFENAC SODIUM IN BULK AND TABLET DOSAGE FORM

Objective: The objective is to study the development of a simple, rapid, specific, precise, and accurate reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of serratiopeptidase (SER) and diclofenac (DC) sodium in bulk and tablet formulation.Methods: RP-HPLC method was developed for the simultaneous estimation of SER and DC sodium in tablet formulation. The separation was achieved by Kromasil C18 column (250 mm × 4.6 mm, 5 μm particle size) with phosphate buffer pH-7 and o-phosphoric acid:methanol:acetonitrile (5:4:1% v/v/v). Flow rate was maintained at 1 mL/min and UV detection was carried at 270 nm.Result: For RP-HPLC method, the retention time for SER and DC sodium was found to be 3.3833 min and 8.1667 min, respectively. The method was validated for accuracy, precision, and specificity. Linearity for SER and DC sodium was in the range of 5–50 μg/ml.Conclusion: The developed RP-HPLC method is simple, accurate, rapid, sensitive, precise, and economic. Hence, this method can be employed successfully for the estimation of SER and DC sodium in both bulk and tablet dosage forms

Determination of Ascorbic Acid Content using the Reverse Phase–High Performance Liquid Chromatography (RP-HPLC) Method

A method of reverse phase-high performance liquid chromatography (RP-HPLC) was evaluated in ascorbic acid determination. The determination was conducted at optimum conditions for RP-HPLC method, including ethanol as mobile phase, flow velocity of 0.8 mL/min, running time of 5 minutes, injection volume of 10 µL, UV detector, wavelength of 245 nm with internal standard of ascorbic acid in metaphosphate-acetic acid. Result of measurement was validated by AOAC Official Method 967.21. It was obtained that RP-HPLC method was high sensitive and precision method due to its limit of detection was 0,5 µg/mL and  RSD  was ≤ 2%. The results of the determination of vitamin C content in fresh ceruring fruit using RP-HPLC method and the titrimetry method namely, 52.9057 ± 0.17 and 54.2066 ± 0.87 mg in 100 g sample, respectively. Based on statistical analysis (t-test), there were no significant differences for the two methods used. The percentage of recovery obtained is excellent in the range of 97.9-104.3%. Therefore, the RP-HPLC method is expected to be an accurate method for routine analysis of vitamin C. Key word: ascorbic acid, reverse phase, HPLC, titrimetric.

Analytical Method Development and Validation for the Estimation of Sugammadex

A simple, precise, accurate, specific RP-HPLC method developed for sugammadex in bulk and simulated mixture. Chromatographic separation is achieved by C18 column (250 x 4.6 mm, 5µ) in isocratic mode. The optimized mobile phase consists of acetonitrile and double distilled water in ratio of 20:80%v/v at a flow rate of 0.5mL/min and sugammadex was monitored at 210nm. Retention time of the drug was found to be 3.39min. The linearity obtained in range of 50 – 250 µg/mL.  %RSD mean for precision and %Recovery mean of the sugammadex were found to be 0.63 and 99.04% - 99.84% respectively. Stability indicating nature of RP-HPLC method was established by applying the degradation condition. The results indicate that developed RP-HPLC method would be suitable for estimation of drug in presence of degradant product. The above developed method was validated according to ICH guideline.  Keywords: Sugammadex, Assay, Simulated Mixture, Forced Degradation, Validation

RP-HPLC method development and validation for the estimation of antifungal drug terbinafine HCL in bulk and pharmaceutical dosage form

In the present work RP-HPLC method has been developed for the quantitative estimation of Terbinafine hydrochloride in bulk drug and pharmaceutical formulations. A rapid and sensitive RP-HPLC Method with PDA detection (220 nm) for routine analysis of in Bulk drug and Pharmaceutical formulation was developed. Chromatography was performed with mobile phase containing a mixture of Potassium dihydrogen phosphate and Acetonitrile (65:35 v/v) with flow rate 1.5 ml/min. The linearity was found to be in the range of 50-150 µg/ml with (r2=0.999). The proposed method was validated by determining sensitivity, accuracy, precision, LOD, LOQ and system suitability parameters according to ICH guidrelines

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR PHENYTOIN SODIUM AND PHENOBARBITONE IN BULK AND PHARMACEUTICAL DOSAGE FORM

Objective: To develop an accurate, simple, rapid, precise, and linear RP-HPLC method for the simultaneous estimation of phenytoin sodium and phenobarbitone in tablet dosage form and validated as per ICH guidelines.Methods: The method used was a reverse phase HPLC (RP-HPLC) method using Hypersil BDS C18, (250×4.6 mm, 5 μm) column, mobile phase comprising of methanol: phosphate buffer (pH 5.0) (50:50), flow rate of 1.0 ml/min and a detection wavelength of 215 nm using a UV detector.Results: The retention time for phenytoin sodium and phenobarbitone was found to be 3.97 min and 6.90 min, respectively. The linearity of developed method was achieved in the range of 10-30 μg/ml for phenytoin sodium and 3-9 μg/ml for phenobarbitone. The detection (LOD) and quantitation (LOQ) limits were 1.44 and 4.36 µg/ml for phenytoin sodium and 0.40 and 1.35 µg/ml for phenobarbitone respectively.Conclusion: A simple, accurate, precise, linear and rapid RP-HPLC method was developed for simultaneous quantitative estimation of phenytoin sodium and phenobarbitone in bulk and pharmaceutical formulation. The method was validated as per ICH guidelines. Hence, the method holds good for the routine analysis of phenytoin sodium and phenobarbitone in various pharmaceutical industries as well as in academics

Benefits and Limitations of Lab-on-a-Chip Method over Reversed-Phase High-Performance Liquid Chromatography Method in Gluten Proteins Evaluation

RP-HPLC (reversed-phase high-performance liquid chromatography) is widely used to determine the amounts of the different gluten protein types. However, this method is time-consuming, especially at early stages of wheat breeding, when large number of samples needs to be analyzed. On the other hand, LoaC (Lab-on-a-Chip) technique has the potential for a fast, reliable, and automatable analysis of proteins. In the present study, benefits and limitations of Lab-on-a-Chip method over RP-HPLC method in gluten proteins evaluation were explored in order to determine in which way LoaC method should be improved in order to make its results more compliant with the results of RP-HPLC method. Strong correlation (P lt = 0.001) was found between numbers of HMW glutenin peaks determined by LoaC and RP-HPLC methods. Significant correlations (P lt = 0.05) were obtained between percentages of HMW and LMW glutenin subunits calculated with regard to total HMW + LMW area. Even more significant correlation (P lt = 0.001) was found when percentages of individual HMW areas were calculated with regard to total HMW. RP-HPLC method showed superiority in determination of gliadins since larger number and better resolution of gliadin peaks were obtained by this method

ANALYSIS OF LYNESTRENOL IN HUMAN PLASMA IN VITRO BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY UV-VIS: EUROPEAN MEDICINES AGENCY GUIDELINE

Objective: Development and validation of reverse phase high performance liquid chromatographic (RP-HPLC) method with UV-Vis detector for in vitro determination of lynestrenol with levonorgestrel as an internal standard in human plasma. Methods: The RP-HPLC method was developed using a C18 Sunfire© waters column with a mobile phase of acetonitrile containing 0.1% formic acid in water (60:40), respectively, at a flow rate of 1.0 ml/min and was detected at a wavelength of 204 nm. Lynestrenol and levonorgestrel were extracted from human plasma using pentane with protein precipitation method. Results: The RP-HPLC method was able to selectively quantify lynestrenol in blood plasma on 40 ng/ml. The assay exhibited a linear dynamic range 40-1000 ng/ml for lynestrenol with retention time 4.0 second, and the coefficient correlation (r) was 0.9994. Accuracy (% diff) of this method was-10.81% to 8.72% with precision (CV) being 3.84% to 8.12%, and complete recovery was established to be 98.27% to 106.49%. The method was sensitive, selective, and has simple sample preparation extraction lynestrenol in plasma with pentane was successfully developed. Conclusion: The method can be used to analyze lynestrenol in blood plasma, with a simple pretreatment procedure using pentane

Validation of Stability Indicating Method and Degradation Kinetic Study of Apremilast

A novel stability indicating RP- HPLC method was developed for the estimation of Apremilast in bulk and marketed formulation. Separation was achieved by using Shimadzu HPLC Analytical Technologies Limited C18 (250 mm x 4.6 mm, 5µm) as stationary phase. The optimized mobile phase consist of potassium dihydrogen ortho phosphate (pH-3.2): acetonitrile in ratio of 40:60 %v/v with flow rate of 1mL/min by using methanol as diluent. Retention time of Apremilast was found to be 5.4 min which was estimated at wavelength 360nm. Linearity of Apremilast was observed in the concentration range of 50-400µg/mL with r² value of 0.9999. Assay of Apremilast tablet was found to be 99.14-100.75%. Stability indicating nature of RP- HPLC method was estimated by conducting degradation kinetic study. The forced degradation of Apremilast bulk indicate that degradation in acidic, alkali, oxidative and photolysis condition were found to be 21%, 6.5%, 25.7% and 3.9% respectively. The kinetic study of apremilast in alkali degradation followed first order kinetic study. The result indicate that the developed RP-HPLC method is suitable for estimation of Apremilast in presence of degradant product. The above method was validated as per ICH guideline. Keywords: Apremilast, RP-HPLC, Validation, Forced Degradation Study, Alkali Degradation Kinetic stud